The invention involves viral vectors that can be used to transduce a target cell, i.e., to introduce genetic material into the cell. The targets of interest are eukaryotic cells and particularly human cells. The transduction can be done in vivo or in vitro. More particularly the invention concerns viral vectors that have chimeric envelope proteins and contain the IgG-binding domain of protein A. These vectors when used in conjunction with antibodies targeting a particular cell are particularly useful for gene therapy.
A variety of viral based vectors have been employed to transfer and to express a gene of interest into a eukaryotic target cell. Recombinant DNA techniques are used to replace one or more of the genes of the virus with the gene of interest operably linked to a promoter that is functional in the target cell. The construct, termed a viral vector, infects the target cell, using the physiological infective xe2x80x9cmachineryxe2x80x9d of the virus, and expresses the gene of interest instead of the viral genes. Because not all the genes of the virus are present in the vector, infection of the target by the vector does not produce viral particles. Viruses that have been used to infect human or mammalian target cells include herpes virus, adenovirus, adeno-associated virus and derivatives of leukemia-type retroviruses. Among the retroviruses of particular interest in the transduction of cells of human origin are constructs based on amphotropic retroviruses.
Retroviruses are particularly well suited for transduction of eukaryotic cells. The advantages of a vector based this type of virus include its integration into the genome of the target cell so that the progeny of the transduced cell express the gene of interest. Secondly, there are well developed techniques to produce a stock of infectious vector particles that do not cause the production of viral particles in the transduced target cell. Lastly, the production and purification of stocks vector particles having titers of 106 TCIU/ml can be accomplished.
One disadvantage of the use of retroviral vectors is that there is presently no practical general, method whereby a particular tissue or cell type of interest can be specifically transduced. Previous efforts to this end have included surgical procedures to limit to specific organs the physical distribution of the viral vector particles (Ferry, N. et al., 1991, Proc. Natl. Acad. Sci. 88:8377). Alternatively, practitioners have taken advantage of the fact that type C retroviruses only infect dividing cells. Thus, a population of cells, e.g., bone marrow cells, was removed from a subject and cultured ex vivo in the presence of growth factors specific for the specific target cell which, thus, comprises most of dividing cells in the culture. See, e.g., Wilson, J. M. et al., 1990, Proc. Natl. Acad. Sci. 87:439-47; Ohashi, T. et al., 1992, Proc. Natl. Acad. Sci. 69:11332-36. After transduction the dividing cells must be harvested and, 30 for many purposes, reimplanted into the subject. The technical difficulties of the ex vivo culture technique combined with the unavailability of growth factors of specific for some types of cells have limited the application of this approach.
A second difficulty presented by the use retroviral based vectors is that a recombination may occur between sequences of vector and an endogenous retrovirus. Such recombination can give rise to a replication competent virus that can cause the production of infectious particles by the target cell. In contrast to herpes virus or adenovirus infection, retroviral infections are not necessarily self-limiting.
Notwithstanding these difficulties, retrovirus vectors based on amphotropic murine leukemia retroviruses that infect human cells, have been approved for use in human gene therapy of certain diseases, for example adenosine deaminase and low density lipoprotein receptor deficiencies and Gaucher""s Disease. See, e.g., Miller A. D., 1992, Nature 357:455; Anderson, W. F., 1992, Science 256:808; and Crystal, R. G., 1995, Science 270:404-410.
One approach to overcoming the limitations of using amphotropic retrovirus vectors in human cells has been to mutate the gene encoding the protein on the viral surface that determines the specificity of infection of the virus, the gp70 protein. Using recombinant DNA technology a xe2x80x9cmutantxe2x80x9d virus is constructed that has had small regions of the gp70 sequence replaced by predetermined sequences. The limits of this approach are set by the requirement for knowledge of the sequence that will enable infection of the target of interest. However, when this knowledge was available, the anticipated alteration in viral specificity has been observed (Valsesia-Wittmann, S., 1994, J. Virol. 68:4609-19).
Retrovirus vectors are the most efficient tools available today to stably transduce genes into the genomes of vertebrate cells. Murine leukemia retrovirus (MLV)-based vectors commonly used for gene transfer are classified on the basis of their host range as either ecotropic or amphotropic. Murine ecotropic virions can only infect mouse or rat cells, but murine amphotropic viruses can infect cells of most species, including human cells. Because of their ability to infect such a broad spectrum of cells, a major drawback to the use of amphotropic virus vectors is the fact that these vectors lack target-cell specificity.
Several attempts to alter the host range of retroviruses have been reported to date. Recently, direct modifications of the envelope protein of murine leukemia virus (MLV) have been shown to redirect the viral tropism. A recombinant virus containing a fragment encoding a single Fv antibody chain at the N terminal region of the MLV env gene has been shown to recognize the corresponding epitopes and infect human cells (Russell, S. J. et al., 1993, Nucleic Acids Res. 21:1081-1085; Somia, N. V. et al., 1995, Proc. Natl. Acad. Sci. USA 92:7570-7574; Marin, M. et al., 1996, J. Virol. 70:2957-2962). Kasahara et al. have made a chimeric ecotropic virus containing an erythropoietin-envelope fusion protein (Kasahara, N. et al., 1994, Science 266:1373-1376). This chimeric virus has been shown to infect human cells bearing the erythropoietin receptor. However, this type of approach suffers from at least two limitations. First, each targetable vector must be constructed de novo. It is unlikely:that the incorporation of different targeting elements in the envelope of the virus can always be achieved with equal success and without reducing the virus titers than can be obtained. Second, virions constructed to directly bind to specific targets in human cells are intrinsically unsafe, as wild-type recombinants could produce potentially harmful effects patients treated with such vectors. By contrast, virions constructed as outlined in this manuscript are uninfectious to human cells in the absence of an accompanying targeting reagent, such as a mAb, which is produced separately and only provided in conjunction with the virus at a convenient time.
An alternative to altering the specificity of binding of the gp70 protein itself is to employ a second, novel structure that binds or is bonded to both the viral particle and to the target cell. In one example of this approach, lactose molecules were covalently coupled, by a non-specific reaction, to the envelope proteins of an ecotropic retrovirus, which does not normally infect human cells. A human hepatocellular carcinoma that was known to have receptors for lactose-containing proteins was found to be susceptible to transduction by this vector complex, although the integration of the transduced gene of interest in the target cell chromosome was not directly demonstrated (Neda, H. et al., 1991, J. Biol. Chem. 266:14143). No evidence of expression was observed in a hepatocellular carcinoma that lacked the lactose specific receptor. The method of Neda results in a variable number of binding sites for the exposed acceptor-on the target cell, attached to each derivatized or bound envelope protein and, of course, is limited to the case wherein the target cell has a lactose receptor.
Another approach to targeting is the use of adapter molecules involved an adapter that was not covalently coupled to the vector. The use of this type of adapter has been attempted by Roux and his colleagues, who have published several reports that relate to this strategy (Patent Publication FR 2,649,119 to Piecheczyk, Jan. 4, 1991; Roux P. et al., 1989, Proc. Natl. Acad. Sci. 86:9079-83; Etienne-Julan, M. et al., 1992, J. Gen. Virol. 73:3251-55). Roux and colleagues have constructed adapters from two types of proteins, both typically antibodies, by biotinylating the proteins and utilizing avidin or streptavidin tetramer, a protein which binds four biotin molecules, to form aggregates of up to four of the biotinylated proteins.
A better approach is described in U.S. Pat. No. 5,753,499, Meruelo et al., the contents of which are hereby incorporated by reference into this patent application. Meruelo et al. describe viral complexes and methods of use to prepare pre-formed adaptors and linkers suitable for gen therapy. They are particularly well-suited for retroviral systems.
Sindbis virus, a member of the Alphavirus genus, has received considerable attention for use as virus-based expression vectors. Many properties of alphavirus vectors make them a desirable alternative to other virus-derived vector systems being developed, including rapid engineering of expression constructs, production of high-titered stocks of infectious particles, infection of nondividing cells, and high levels of expression (Strauss, J. H. et al., 1994, Microbiol. Rev. 58:491-562; Liljestrxc3x6m, P. et al., 1991, Biotechnology 9:1356-1361; Bredenbeek, P. et al., 1992, Semin. Virol. 3:297-310; Xiong, C. et al., 1993, Science 243:1188-1191). However, a major drawback to the use of Sindbis virus vectors is the fact that these vectors lack target-cell specificity. For mammalian cells, at least one Sindbis virus receptor is a protein previously identified as the high-affinity laminin receptor, whose wide distribution and highly conserved nature may be in part responsible for the broad host range of the virus (Strauss, J. H. et al. 1994; Wang, K. -S. et al., 1992, J. Virol. 66:4992-5001). It is desirable to alter the tropism of the Sindbis virus vectors to permit gene delivery specifically to certain target cell types. This will require both the ablation of endogenous viral tropism and the introduction of novel tropism. In the mature Sindbis virus virion, a plus-stranded viral genome RNA is complexed with capsid protein C to form icosahedral nucleocapsid that is surrounded by lipid bilayer in which two integral membrane glycoproteins, E1 and E2 are embedded (Strauss, J. H. et al., 1994). Although E1 and E2 form heterodimer that functions as a unit, the E2 domain appears to-be particularly important for binding to cells. Monoclonal antibodies (mAbs) capable of neutralizing virus infectivity are usually E2 specific, and mutations in E2, rather than E1, are more often associated with altered host range and virulence (Stanley, J. et al., 1985, J. Virol. 56:110-119; Olmsted, R. A. et al., 1986, Virology 148:245-254; Polo, J. M. et al., 1988, J. Virol. 62:2124-2133; Lustig, S. et al., 1988 J. Virol. 62:2329-2336). Recently, a Sindbis virus mutant was identified which contained an insertion in E2 and exhibited defective binding to mammalian cells. This mutant is expected to be useful for development of targetable Sindbis virus vectors (Dubuisson, J. et al., 1993, J. Virol. 67:3363-3374).
Grieve et al. (International Publication No. WO 94/17813 published Aug. 18, 1994, xe2x80x9cDefective Sindbis Virus Vectors That Express Toxoplasma Gondii P30 Antigensxe2x80x9d) report the use of defective sindbis viral vectors to protect mammals from protozoan parasites, helminth parasites, ectoparasites, fungi, bacteria and viruses, the contents of which are hereby incorporated by reference. Garoff et al. (International Publication No. WO 92/10578 published Jun. 25, 1992, xe2x80x9cDNA Expression Systems Based On Alphavirusesxe2x80x9d) describe the use of alphaviruses to express protein sequences for immunization or protein production, the contents of which are hereby incorporated by reference. Davis et al. (U.S. Pat. No. 5,185,440 issued Feb. 9, 1993, entitled xe2x80x9ccDNA Clone Coding For Venezuelan Equine Encephalitis [(VEE)] Virus And Attenuating Mutations Thereof) disclose cDNA encoding VEE and methods of preparing attenuated Togaviruses, the contents of which are hereby incorporated by reference. Huang et al. (U.S. Pat. No. 5,217,879 issued Jun. 8, 1993, entitled xe2x80x9cInfectious Sindbis Virus Vectorsxe2x80x9d) describe infectious Sindbis virus vectors with heterologous sequences inserted into the structural region of the genome, the contents of which are hereby incorporated by reference. Schlessinger et al. (U. S. Pat. No. 5,091,309 issued Feb. 25, 1992, entitled xe2x80x9cSindbis Virus Vectorsxe2x80x9d) describe RNA vectors based on the Sindbis Defective Interfering (DI) particles with heterologous sequences inserted, the contents of which are hereby incorporated by reference. Dalemans et al. (International Publication No. WO 95/27069 published Oct. 12, 1995, xe2x80x9cAlpha Virus RNA As Carrier For Vaccinesxe2x80x9d) report the medical use of alphaviruses, specifically the Semliki Forest Virus, to delivery exogenous RNA encoding a antigenic epitope dr determinant, the contents of which are hereby incorporated by reference. Dubensky et al., International Publication No. WO 95/07994 published Mar. 23, 1995, xe2x80x9cRecombinant Alphavirus Vectorsxe2x80x9d describe recombinant retroviral alphavirus vectors for delivery of heterologous genes to target cells, the contents of which are hereby incorporated by reference. Sjxc3x6berg et al., International Publication No. WO 95/31565 published Nov. 23, 1995, xe2x80x9cAlphavirus Expression Vectorxe2x80x9d disclose vectors for enhanced expression of heterologous sequences downstream from an alphavirus base sequence, the contents of which are hereby incorporated by reference. Liljestrxc3x6m et al., International Publication No. WO 95/27044 published Oct. 12, 1995, xe2x80x9cAlphavirus cDNA Vectorsxe2x80x9d describe a cDNA construct that may be introduced and transcribed in animal or human cells, the contents of which are hereby incorporated by reference.
The invention concerns viral vectors and their use. Specifically, the invention is concerned with viruses having a protein on the viral particle surface that is a chimeric protein comprising a viral envelope protein and an IgG-binding domain of protein A. Because protein A binds to an Fc region of antibody, these chimeric proteins enable one to use an antibody to target the viral particle to a desired cell to which the antibody binds and not to a cell to which the antibody does not bind.